BrAcFsk, an alkylating derivative of forskolin was used to probe the forskolin binding sites on bovine brain adenylyl cyclase and the human erythrocyte glucose transporter. An immunoblotting procedure was developed to detect the incorporation of BrAcFsk into proteins which involved Western blotting of BrAcFsk-labeled proteins followed by detection with anti-forskolin antiserum and I-125 labeled protein A. Preactivated adenylyl cyclase was purified by elution from forskolin affinity columns with BrAcFsk. The proteins which had covalently incorporated BrAcFsk were identified by the immunoblotting procedure. BrAcFsk specifically covalently labeled the-120 kDa protein which was purified on the forskolin affinity resin suggesting that BrAcFsk was reacting with reactive nucleophilic group(s) at the forskolin binding site. The 120 kDa protein was phosphorylated by cAMP-dependent protein kinase and contained N-linked oligosaccharides consistent with this protein being the adenylyl cyclase catalytic subunit. No significant incorporation of BrAcFsk into the erythrocyte glucose transporter was detected by Western blot analysis with anti-forskolin antiserum. Thus, in contrast to the adenylyl cyclase, there is no reactive nucleophilic group at the forskolin binding site on the erythrocyte glucose transporter. Alkylating derivatives of forskolin are being used to study other forskolin binding proteins.